Gentamicin Ototoxicity Requires Functional Mechanotransducer Channels

Objective: Test the hypothesis that open mechanotransducer (MET) channels are required for aminoglycoside entry and toxicity in auditory hair cells. Method: Cochlear cultures from postnatal 4-day-old rats were treated with gentamicin with and without MET channel blockers. The effects of MET channel blockers on hair cell survival were assessed by cell counts after immunohistochemistry. Two-photon imaging was used to quantify uptake of Texas-Red-conjugated gentamicin (GTTR) into live hair cells. Results: Gentamicinalone (0.1mM) caused 76.5% hair cell loss in the basal cochlear turn. Cotreatment with MET channel blockers, 1.0 mM curare, and 0.5 mM quinine conferred protection (0.4% and 7.4% hair cell loss, both P < .001); whereas treatment with endocytosis inhibitors, 2.6 μM conconavalin A and 80 µM dynosore, did not (63.7% and 76.0% hair cell loss). Two-photon imaging showed that GTTR uptake (3 µM) into live hair cells was rapid and selective. Quinine and curare, but not concanavalin A, reduced GTTR uptake. Furthermore, as low calcium increases the open probability of MET channels, hypocalcemia showed facilitated GTTR uptake. Conclusion: Functional MET channels are required for aminoglycoside uptake into hair cells and its toxicity. Results suggest that limiting permeation of aminoglycosides through MET channels is a potential therapeutic target for preventing hair cell death.